Detecting interactions between molecules forms the basis of many analytical methods. The interaction can be detected and quantified through a number of schemes, e.g. precipitation, separation or through different marker molecules or reactions. Such an example is the development of immunoassays during the last three decades, which has revolutionized determination of drugs and hormones in clinical and pharmaceutical chemistry as well as contaminants in the environmental area. Almost all immunomethods require labels attached either to the antibody or the antigen. Another example is the binding between a DNA-probe and its complementary DNA-strand or DNA-fraction. A number of receptors or the complementary molecule can be studied using the same approach.
There are a number of disadvantages associated with labels. It they are radioactive the work has to be carried out under strict safety regime and handling of waste is costly. The use of enzymes as labels requires an additional time-consuming incubation step. Common for all labels are that they require a synthetic coupling to either an antigen or an antibody or generally to the recognition element or the analyte. A big label may change the affinity between the molecules which is of particular concern when an assay is performed by. competition between an analyte from the sample and an added labeled molecule. Many affinity interactions cannot be studied because of this. Recognition of DNA-binding through the use of electrochemical intercalators shows low sensitivity. Many attempts have therefore been made to detect the binding itself by potentiometric [Taylor, R. F.; Marenchic, I. G.; Spencer, R. H. Anal. Chim. Acta 1991, 249, 67-70], piezoelectric [Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336], or optical measurements [Lxc3x6fxc3xa5s, S. Pure Appl. Chem. 1995, 67, 829-834].
Attempts have previously been made to use capacitance measurements for detecting molecular interactions without the use of labels. A molecule with affinity for the analyte should be immobilized on a conducting electrode surface so that it can interact with the analyte in solution in such a way that the interaction causes a change in capacitance. This principle has been used in immunochemistry, by immobilization to oxide surfaces [Bataillard, P.; Gardies, F.; Jaffrezic-Renault, N.; Martelet, C.; Colin, B.; Mandrand, B. Anal. Chem. 1988, 60, 2374-2379] or for recognition of DNA-sequences [Souteyrand, E.; Martin, J. R.; Cloarec, J. P.; Lawrence, M. Eurosensors X, The 10th European Conference on Solid-State Transducers, 1996, Leuven, Belgium].
Self-assembled monolayers of thiols, sulfides and disulfides on gold electrodes have been widely studied and long-chain alkanethiols are known to form insulating well-organized structures on gold substrates [Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc 1987, 109, 3559-3568]. The binding formed between the sulphur atom and gold is very strong and the formed self-assembled monolayers (SAM""s) are stable in air, water and organic solvents at room temperature [Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335]. It has been suggested that microcontact printing [Mrksich, M.; Whitesides, G. M. Tibtech 1995, 13, 228-235] and photolithography [Bhatia, S. K.; Hickman, J. J.; Ligler, F. S. J. Am. Chem. Soc. 1992, 114, 4432-4433] can be used to pattern surfaces with functionalized self-assembled monolayers for biosensor production with low cost for a diversity of applications, but until now it has not been possible to produce direct affinity sensors with high sensitivity.
Terrettaz et al, Langmuir 1993, 9, 1361-1369, discloses a sensor, e.g. for assaying cholera toxin, where the ganglioside GM1 has been bound to a SAM layer. The detection limit for capacitance measurements using the sensor is somewhere within the range from 10xe2x88x926 to 10xe2x88x929 M. The article states that capacitance measurements are unsuitable for assaying cholera toxin because the capacitance changes were too small, and hence, the sensitivity is too low.
Self-assembled monolayers of thiols on gold, with antigenic terminating groups have been reported before, but they had coverages of only 14, 19 or 31% for different electrodes [Taira, H.; Nakano, K.; Maeda, M.; Takagi, M. Anal. Sci. 1993, 9, 199-206]. The lowest measured value in the article was at an antibody concentration of 10 ng/ml, which can be compared to 1 pg/ml of antigen measured with our invention (See Example 1). The higher sensitivity obtained with our electrode can be explained by that the gold surface is first covered with a self-assembled monolayer of a thiol, sulphide or disulphide giving a high coverage of the surface, therafter the recognition element is immobilized on the surface and as the last step the surface is plugged with another thiol. The saturation seems to occur at similar concentrations in the two cases if the larger bulk of the antibody compared to the antigen is taken into account. This comparison thus supports the arguments given above that a dense layer is of great importance for a high sensitivity.
DNA-probes have been immobilized e. g to SiO2 and a sensitivity of 10 ng/ml was obtained [Souteyrand, E.; Martin, J. R.; Cloarec, J. P.; Lawrence, M. Eurosensors X, The 10th European Conference on Solid-State Transducers, 1996, Leuven, Belgium].
A peptide bound to an alkylthiol was also immobilized as a self-assembled layer on gold, but the antibody concentration was in this case in the mg/ml range making it a less succesful sensor [Rickert, J.; Wolfgang, G.; Beck, W.; Jung, G.; Heiduschka, P. Biosens. Bioelectron. 1996, 11, 757-768].
One of these previous approaches are illustrated in the patent EP 244326. The recognition element is bound to an insulating layer on top of a conducting substrate, the insulating layer typically being an oxide. The oxide layer has to be thick, typically 70 nm on silicon, in order to be stable and sufficiently insulating, resulting in a low sensitivity. It is difficult to obtain good surface coverage on oxides and the recognition elements are not well ordered.
Rojas, M.; Kxc3x6niger, R.; Stoddart, F.; Kaifer, A.; J. Am. Chem. Soc. 1995, 117, 336-343 discloses an assay method for determining ferrocene in a sample using cyclodextrin. All hydroxy groups of cyclodextin are substituted by thiol groups, and the modified cyclodextrins are chemically adsorbed to a gold surface. Empty spaces on the gold surface between the adsorbed modified cyclodextrin molecules are filled with adsorbed pentanethiols. The lowest ferrocene concentration determined is 5 xcexcM.
There is always a need for improvements of analysis techniques. Especially when assaying biochemical compounds it is often necessary to be able to determine concentrations below 1 ng/ml.
It has now turned out that unexpectedly good capacity affinity sensors, suitable for determining the presence of a certain compound of interest by capacitance measurements using an electrode which can be produced by a method comprising the steps of:
a) providing a piece, of a noble metal where said piece optionally can be a rod or, alternatively a piece of insulating material such as glass, silica or quartz, on which a noble metal is sputtred or printed;
b) providing a first SAM-forming molecule comprising a coupling group and/or an affinity group specifically binding said compound of interest;
c) contacting the piece in step a) with the first SAM-forming molecule in step b), thereby obtaining a self-assembling monolayer on said noble metal surface;
d) in case the first SAM-forming molecule does not comprise an affinity group, contacting said self-assembling monolayer on said noble metal piece with an affinity molecule specifically binding said compound of interest, thereby coupling the affinity molecule to the self-assembling monolayer; and
e) contacting the piece obtained in step c) or d) with a second SAM-forming molecule, thereby obtaining a noble metal surface that is at least 90%, preferably at least 97% covered with a self-assembling monolayer.
The detection limits reported in this invention are at least three orders of magnitude better than those reported previously for capacitive immunosensors and a comparison is therefore necessary in order to explain why this invention succeeds so exceptionally well. The insights behind this invention are that the recognition layer must be thin, well-ordered and it must cover at least 90%, preferably at least 95%, more preferably at least 97%, and most preferably at least 99% of the sensor surface. In a subsequent step, any free spots between the recognition elements are xe2x80x9cpluggedxe2x80x9d, i.e. covered with a second self-assembling monolayer-forming molecule, e.g. an alkanethiol comprising 3-25 carbon atoms preferably in a straight chain, after obtaining a self-assembling monolayer comprising affinity groups, thereby increasing the tightness and insulation. A capacitive biosensor is covered by an immobilized layer with the recognition element toward the solution. Electrically it is equivalent to a capacitor between the conducting metal electrode and the conducting solution. Another layer forms when a molecule binds to the recognition element thereby replacing aqueous solution with a non-conducting organic molecule. This is equivalent to the formation of an additional capacitor in series with the first, thereby decreasing the total capacitance.
Any part of the surface that allows the aqueous solution to penetrate below the plane where the recognition takes place will act like a short-circuiting element. The capacitance will therefore increase due to the higher dielectric constant of the penetrating aqueous solution. Oxide layers are not well ordered and it is therefore impossible to form a dense recognition layer. Self-assembled monolayers are much better ordered and a more perfect coverage can therefore be expected in the immobilized layers. Furthermore the self-assembled monolayers are much thinner than the oxide layers, resulting in a larger capacitance in series with the capacitance formed when molecules bind on the surface. This makes it easier to detect changes in the capacitance when an analyte binds to the surface.
This invention describes an capacity affinity sensor based on measurements of the capacitance change at conducting surfaces. The grafted recognition layer should be electrically insulating to prevent interferences from redox couples in the electrolyte solution and high Faradaic background currents. On the other hand, it should be as thin as possible in order to achieve high sensitivity. The use of self-assembled binding to gold or other noble metals gives especially thin and compact layers. The invention also shows how additional insulation can be obtained by plugging with a different type of self-assembling molecule.
Accordingly, the present invention relates to a method for producing a capacity affinity sensor, wherein a piece of a noble metal is covered with a layer of a self-assembling monolayer-forming molecule comprising coupling groups. Affinity molecules are then coupled to these self-assembling monolayer-forming molecules. Subsequently any remaining free spots on the noble metal surface is covered by a second self-assembling monolayer-forming molecule.
In another aspect, the present invention relates to a capacity affinity sensor comprising a noble metal piece substantially completely covered with a self-assembling monolayer comprising first and second self-assembling monolayer-forming molecules, and where affinity molecules that specifically binds to a certain molecule of interest have been coupled to the first self-assembling monolayer-forming molecules.
In yet another aspect, the present invention relates to a method for qualitatively or quantitatively determining the presence of a certain compound of interest. A capacity affinity sensor, comprising a noble metal piece substantially completely covered with a self-assembling monolayer comprising first and second self-assembling monolayer-forming molecules, and where affinity molecules that specifically binds to a certain molecule of interest have been coupled to the first self-assembling monolayer-forming molecules, is contacted with a liquid sample comprising the compound of interest and the sensor""s capacitance is determined.
In a further aspect, the present invention relates to using said sensors for analysing certain compounds of interests, such as human chorionic gonadotropin hormone (HCG), interleukin-2, human serum albumin, atrazine or a certain DNA sequence.
Definitions
As disclosed herein, the terms xe2x80x9cself-assembled monolayerxe2x80x9d and xe2x80x9cSAMxe2x80x9d are synonyms and relates to the spontaneous adsorption of film components from a solution onto a solid surface making a well-ordered monolayer. Such a layer on gold substrates have previously been described substrates [Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc 1987, 109, 3559-3568].
As disclosed herein, the term xe2x80x9cnoble metalxe2x80x9d relates to a metal chosen from the group of gold, silver, copper, platinum and palladium. Gold is preferred.
As disclosed herein, the term xe2x80x9caffinity moleculexe2x80x9d relates to a molecule which specifically binds to a certain molecule of interest. If the molecule to be determined is an antigen, the affinity molecule might be an antibody, preferably a monoclonal antibody, or an antibody fragment such as a F(abxe2x80x2)2 fragment. If a certain nucleic acid sequence is to be identified, the affinity molecule might be a nucleic acid probe specifically hybridizing to said nucleic acid sequence. The present invention can also be used in relation to affinity-mediating biomolecules in general, for example in situations where certain nucleic acids bind to antigens other than nucleic acids, such as proteins. The skilled person is well aware of how to choose suitable affinity molecules for a certain compound to be determined.
As disclosed herein, the term SAM-forming molecule relates to a molecule having the ability of forming a self-assembling monolayer on a noble metal. A SAM-forming molecule comprises at least one thiol, sulphide or disulphide group and may optionally also comprise an affinity group. Affinity molecules are coupled to small SAM-forming molecules comprising coupling groups in a separate step Examples of such small SAM-forming molecules comprising coupling groups are thioctic acid and cysteamine. This coupling step is carried out after formation of the self-assembling monolayer on the noble metal surface. The skilled person is well aware of how to choose suitable coupling reactions and coupling groups. In the following examples, a self-assembling monolayer consisting of thioctic acid is activated by 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide. Subsequently, an affinity molecule is coupled to the activated monolayer. However, other similar coupling reactions are described in the literature.
As disclosed herein, the term xe2x80x9cpluggingxe2x80x9d refers to treatment in a solution containing a thiol, sulphide or disulphide after immobilization of the affinity molecule to a self-assembling monolayer on a noble metal surface in order to block any unblocked spots on said surface. As already mentioned, it is necessary that the noble metal surface is as completely covered by a SAM as possible in order to optimize the sensitivity of the sensor. Suitable examples of SAM-molecules that can be used for plugging are thiols comprising 3-25 carbon atoms in a straight satured chain. Such SAM-molecules lack coupling groups. A preferred example is 1-dodecanethiol.
As disclosed herein, SCE stands for the saturated calomel electrode; Potentiostatic perturbation means a fast change in potential; HCG stands for human chorionic gonadotropin; IL-2 stands for interleukin 2 and HSA stands for human serum albumin.
The interactions that can be measured using this capacitance sensor includes antigen-antibody, hapten-antibody, peptide-protein, nucleic acids, lectin-hydrocarbon-containing parts, biotin-streptavidin-avidin, receptors-agonist-antagonist, ligand-cells. Complexes can be one part of the affinity pair, e. g. hapten-antibody binding to immobilized hapten as recognition element. Fragment, e. g. of antibodies can be used instead of the native antibody. Recognition element as used in here constitutes any one of the pairs or complexes mentioned above which is immobilized on the electrode surface. Analyte is the molecule to be determined and is normally the other part than the recognition element in the pairs above.
In this invention a solution containing the molecules, complexes or cells to be determined is allowed to make contact with a surface containing the affinity group, after which the capacitance or impedance change when an interaction takes place is determined . The capacitance change takes place between the solution and a metal surface, consisting of solid metal or metal sputtered or printed on an underlaying non-conducting surface. Faradaic reactions with the metal as well as background currents are blocked by the affinity group on the surface, eventually improved by treatment with auxiliary compounds which improve the insulation. The affinity group is bound to the metal surface, either directly through self-assembly, or by binding it to a self-assembled compound on the electrode. It can also be bound through adsorption, polymerization or coating. Measurements are made using electrochemical perturbations followed by recording of the resulting response. The perturbations used in the examples described below are potentiostatic steps or pulses which give rise to current transients from which the capacitance is evaluated. Perturbations can also be amperometric steps in which case the change in potential is used for capacitance evaluation. Perturbations with sinusoidal or other wave-forms have been reported in the litertature. The sensitivity can be improved by allowing a solution containing a secondary specific ligand to bind to the analyte already on the surface, thereby increasing the size of the bound aggregate and the capacitance change.